Effects of antibiotic treatment and phagocyte infiltration on development of Pseudomonas aeruginosa biofilm-Insights from the application of a novel PF hydrogel model in vitro and in vivo.

Background and purpose: Bacterial biofilm infections are major health issues as the infections are highly tolerant to antibiotics and host immune defenses. Appropriate biofilm models are important to develop and improve to make progress in future biofilm research. Here, we investigated the ability of PF hydrogel material to facilitate the development and study of Pseudomonas aeruginosa biofilms in vitro and in vivo. Methods: Wild-type P. aeruginosa PAO1 bacteria were embedded in PF hydrogel situated in vitro or in vivo, and the following aspects were investigated: 1) biofilm development; 2) host immune response and its effect on the bacteria; and 3) efficacy of antibiotic treatment. Results: Microscopy demonstrated that P. aeruginosa developed typical biofilms inside the PF hydrogels in vitro and in mouse peritoneal cavities where the PF hydrogels were infiltrated excessively by polymorphonuclear leukocytes (PMNs). The bacteria remained at a level of ~106 colony-forming unit (CFU)/hydrogel for 7 days, indicating that the PMNs could not eradicate the biofilm bacteria. ß-Lactam or aminoglycoside mono treatment at 64× minimal inhibitory concentration (MIC) killed all bacteria in day 0 in vitro biofilms, but not in day 1 and older biofilms, even at a concentration of 256× MIC. Combination treatment with the antibiotics at 256× MIC completely killed the bacteria in day 1 in vitro biofilms, and combination treatment in most of the cases showed significantly better bactericidal effects than monotherapies. However, in the case of the established in vivo biofilms, the mono and combination antibiotic treatments did not efficiently kill the bacteria. Conclusion: Our results indicate that the bacteria formed typical biofilms in PF hydrogel in vitro and in vivo and that the biofilm bacteria were tolerant against antibiotics and host immunity. The PF hydrogel biofilm model is simple and easy to fabricate and highly reproducible with various application possibilities. We conclude that the PF hydrogel biofilm model is a new platform that will facilitate progress in future biofilm investigations, as well as studies of the efficacy of new potential medicine against biofilm infections.

Solar energy harvesting in the epicuticle of the oriental hornet (Vespa orientalis).

The Oriental hornet worker correlates its digging activity with solar insolation. Solar radiation passes through the epicuticle, which exhibits a grating-like structure, and continues to pass through layers of the exo-endocuticle until it is absorbed by the pigment melanin in the brown-colored cuticle or xanthopterin in the yellow-colored cuticle. The correlation between digging activity and the ability of the cuticle to absorb part of the solar radiation implies that the Oriental hornet may harvest parts of the solar radiation. In this study, we explore this intriguing possibility by analyzing the biophysical properties of the cuticle. We use rigorous coupled wave analysis simulations to show that the cuticle surfaces are structured to reduced reflectance and act as diffraction gratings to trap light and increase the amount absorbed in the cuticle. A dye-sensitized solar cell (DSSC) was constructed in order to show the ability of xanthopterin to serve as a light-harvesting molecule.

Xanthopterin in the Oriental hornet (Vespa orientalis): light absorbance is increased with maturation of yellow pigment granules.

The Oriental hornet bears both brown and yellow colors on its cuticle. The brown component is contributed by the pigment melanin, which is dispersed in the brown cuticle and provides protection against insolation, while the yellow-colored part contains within pockets in the cuticle granules possessing a yellow pigment. These yellow granules (YG) are formed about 2 days prior to eclosion of the imago, and their production continues for about 3 days posteclosion. Xanthopterin is the main component of the granule and lends it its yellow color. Xanthopterin produces a characteristic excitation/emission maximum at 386/456 nm. Characterization by use of mass spectrometry showed the compound to have a molecular ion of 179, as expected from xanthopterin. Spectroscopic examination of the absorption of an entire stripe of yellow cuticle in the course of its metamorphosis revealed that the absorption steadily increases throughout the process to a maximal level of absorption about 3 days posteclosion. In the absence of the YG, the cuticle is permeable to the passage of all wavelengths within the visible range and to the UV range (290-750 nm) in all age groups of hornets. The newly ecloded hornets depart the nest to engage in activities requiring exposure to insolation only as the process of granule formation terminates, namely, when the layer of YG in the cuticle suffices to absorb all the harmful UV radiation.

Some liver functions in the Oriental hornet (Vespa orientalis) are performed in its cuticle: exposure to UV light influences these activities.

The Oriental hornet Vespa orientalis (Hymenoptera, Vespinae) coordinates its daily activities (e.g. flights out of the nest associated with digging activities and removal of the dug soil from the nest) with the amount of insolation. Thus, the stronger the insolation, the more intense the flight activity and vise versa. The hornet's cuticle bears a few yellow stripes interposed among brown parts of the gastral cuticle. These yellow stripes are composed of two elements, namely, a transparent cuticle and underneath it a layer of yellow granules. When the hornets are exposed to UV light, the layer containing the yellow granules is less active than in hornets kept in the dark. This diminished activity entails a lower production of glucose as well as of several enzymes prevalent also in the liver of mammals, like creatine kinase, alanine aminotransferase, aspartate transaminase. Thus solar irradiation stimulates and produces a change in the metabolic activities of the hornet. The fact that hornets link their flight activity with the insolation leads us to speculate that the sun contributes energetically to the hornet's activity.

Hornet flight activity and its correlation with UVB radiation, temperature and relative humidity.

During the active season, extending from June to October, hornets emerge from their nest in the field in all the daytime hours. In the beginning of the season, when the number of workers is relatively small, the number of exits from the nest is fairly uniform numerically throughout the day. However, with the increase in hornet population from July onwards, the number of workers emerging from the nest entrance around noon (1100-1300 h) is by 1-2 orders of magnitude greater than the number of those emerging in the morning or evening hours. This disparity persists till September or October, at which time the workers revert to behave as in the beginning of the season. It appears, therefore, that in this period hornet activities outside the nest are coordinated with the meteorological conditions, and in this regard, the highest correlation is with the ultra violet B (UVB) radiation level and to a lesser extent with the temperature. Presumably, also, the greater noon-hour activity in the nests of hornets in the field stems from the digging hornets benefiting from the greater availability of solar energy at noon, mainly that of UVB radiation. We assume that the hornets are able to utilize the UVB radiation, but what part of their body is "absorbing" the UVB energy is still a matter of further investigation.

Ontogenesis of the thermogenic center in hornets.

In the Oriental hornet, a thermogenic center is located in its prothorax. The present study attempted to elucidate the development of this organ with age, that is, by following the development of the thermogenic center in the hornet from its pupal stage until several days after eclosion of the imago. To this end, use was made of an infrared camera, with which pictures were taken of the prothorax in hornets at various ages, i.e., several days prior eclosion, 24 h after eclosion, and 48-h posteclosion. The photographic findings established that prior to 48-h posteclosion there was no thermally distinct region or spot in the prothorax, but at about 48 h, such a "hot spot," namely, a point whose temperature is greater than that of the rest of the prothorax, does appear, and its appearance coincides with certain specific nest activities like warming of the pupae. Next, an attempt was made to transplant by allograft the region in the prothorax housing the hot spot. Accordingly, several pupae at 2 days prior to eclosion were subjected to the following procedure; their future prothoracic thermogenic center was excised and so also an equally sized piece of cuticle from the dorsal region of their abdomen, and the two now allografted in exchange, i.e., the piece from the prothorax replacing the abdominal piece and vice versa. The result of this exchange-transplant was studied 48 h after eclosion of the operated hornets and showed disruption of heat formation in the prothoracic site coupled with nonappearance of a hot spot in the abdominal site. As for the functional, intact hot spot in the adult hornet, it is characterized by a high concentration of tracheae, with numerous mitochondria in between them that probably contribute to the heat generation.

Prevention of hyperthermia with silk of the oriental hornet, Vespa orientalis: a hypothesis.

Wasps apparently develop normally even under extreme thermal conditions, including deserts. We deemed it worthwhile to set up an experiment wherein wasp brood combs containing a full gamut of brood ranging from eggs up to pupae and a few adults were kept in an incubator whose temperature was gradually raised to 45 degrees C, and the response of the disparate brood to such warming was photographed via Infra Red camera. The finding of this experiment showed that for open brood (i.e., eggs, larvae at various instars, and empty cells) the temperature was close to the ambient temperature, but in the silk coated pupae, the temperature was lower than the ambient by up to 4 degrees C. This lower temperature was retained for at least 90 min of incubation. For comparison we evaluated the relative contribution of the pupae to the phenomenon, by warming also a vacant, (i.e., a broodless and silkless comb) in parallel to a comb from which the pupae had been extricated but the silk weave retained and left behind. We found that the totally empty comb heated up under these conditions to nearly 110 degrees C, whereas the silk-containing vacant cells only heated up to about 40 degrees C. These finding are discussed from two aspects, namely the importance for wasps to maintain a constant temperature throughout the pupating process, and the manner in which the silk weave contributes to such a goal.

Some qualitative liver functions in hornet yellow cuticle.

In social hornets of sub-family Vespinae there are stripes or areas in the cuticle which are endowed with a bright color (yellow, orange, green) that is different from the customary native brown color. In these brightly colored areas, the middle and bottom layers of the cuticle contain a yellow-colored filling material instead of the ordinary lamellae of which the cuticle is comprised. At high magnification, most of the yellow matter is seen to be made up of yellow granules interlinked by string-like threads. Apart from yellow granules, hemolymph and a surrounding cuticle, these areas are also found to contain glucose, iron and triglycerides as well as enzymes like GOT, GPT, CPK and LDH, in high concentrations that vary considerably from those in vespan hemolymph and whose levels are different under illumination than in the dark. Such high levels of enzymes suggest first an association with liver function activities, and secondly, that the difference in their levels when exposed to light and when subjected to darkness is indicative of the influence of the energy of light upon them.

Exposure to an additional alternating magnetic field affects comb building by worker hornets.

Oriental hornet workers, kept in an Artificial Breeding Box (ABB) without a queen, construct within a few days brood combs of hexagonal cells with apertures facing down. These combs possess stems that fasten the former to the roof of the ABB. In an ABB with adult workers (more than 24 h after eclosion), exposed to an AC (50 Hz) magnetic field of a magnitude of B = 50-70 mGauss, the combs and cells are built differently from those of a control ABB, subjected only to the natural terrestrial magnetic field. The effects of the additional magnetic field consist of (a) 35-55% smaller number of cells and fewer eggs in each comb, (b) disrupted symmetry of building, with many deformed and imperfectly hexagonal cells, and (c) more delicate and slender comb stems.

Dual delivery of VEGF and ANG-1 in ischemic hearts using an injectable hydrogel.

Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. While growth factor therapy is promising, the retention in the highly vascularized myocardium is limited and prevents sustained activation needed for adequate cellular responses. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair and function. VEGF and ANG-1 were incorporated in PF hydrogels and their in vitro characteristics were studied. Acute MI was generated in a rodent model with rats randomly assigned to 4 groups; sham, saline, PF and PF-VEGF-ANG1 (n=10 each group). Saline or hydrogel was injected in infarct and peri-infarct areas of the myocardium. After 4weeks, myocardial function was assessed using echocardiography. Tissue samples were harvested for Hematoxylin and Eosin, Masson Trichrome and capillary staining to assess the extent of fibrotic scar and arteriogenesis. Both VEGF and ANG-1 were released in a sustained and controlled manner over 30days. PF-VEGF-ANG1 treated animals showed the best improvement in cardiac function, highest degree of cardiac muscle preservation, and arteriogenesis. This study demonstrates that PF hydrogels can simultaneously provide mechanical support to attenuate adverse myocardial remodelling, and a pro-angiogenic benefit from the sustained VEGF and ANG1 delivery that culminates in a restorative effect following MI. The utility of this synergistic, biomaterial-based growth factor delivery may have clinical implications in the prevention of post-MI cardiac dysfunction. STATEMENT OF SIGNIFICANCE: Acute myocardial infarction (MI) caused by ischemia is the most common cause of cardiac dysfunction. Here, we demonstrated the use of polyethylene glycol-fibrinogen (PF) hydrogels for sustained dual delivery of vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANG-1) to enhance myocardial repair and function. Treated animals showed the best improvement in cardiac function, highest degree of cardiac muscle preservation, and arteriogenesis. This study demonstrates that PF hydrogels can simultaneously provide mechanical support to attenuate adverse myocardial remodelling, and a pro-angiogenic benefit from the sustained VEGF and ANG1 delivery that culminates in a restorative effect following MI.

Antennal and cephalic organelles in the social wasp Paravespula germanica (Hymenoptera, Vespinae): form and possible function.

This paper deals with hairs and organelles present on the head and antennae of the German wasp, Paravespula germanica, and their possible role in sensing the physical and chemical ambience, as well as in intercommunicating both while in flight outside or in the nest. Via scanning electron microscope photography, we detected on the frons plate of the wasp's head, hairs that were about 300 microm long and comprised the longest hairs on the body of the wasps. Additionally, the two antennae bore along their entire length photoreceptors, placoids, campaniforms, trichoids, and agmons. These organelles are located at high but variable density along the antennal segments. The paper provides the dimensions of each of the mentioned organelles, and discusses the possible functions of the organelles as well as of the hairs on the frons. Photographs taken via atomic force microscope reveal that the epicuticle of the antenna is of two typical shapes; one, bearing both longitudinal stripes as well as transverse bands that are about 1 mum in width, and a second granulated form. Conceivably, the wasp uses the various organelles mentioned to communicate with its mates that are some distance away, somewhat like the use of radar by humans.

Presence of a thermoregulatory hot spot in the prothorax of the large carpenter bee and the bumble bee.

In both the large carpenter bee (Xylocopa pubescens) and the bumblebee (Bombus terrestris), a hot spot was detected in the center of the prothorax on its dorsal-external aspect. In both cases, the temperature in this hot spot was found to be greater than the ambient temperature and that at the tip of the gaster. In B. terrestris, it was higher by 9-10 degrees C from that at the gaster tip and by 15-16 degrees C from the ambient temperature, while in X. pubescens the corresponding differences were 11-20 degrees C and 18-19 degrees C, respectively. The recorded thermal differences were not fixed but were rather variable, temporally as well as individually, but invariably all individuals measured showed these temperature differences. Furthermore, in none of the studied specimens was a hot spot detected in any part of the body other than the prothorax. From this hot spot in the prothorax, there is a cascade of temperatures in both directions, that is, anteriorly towards the head and posteriorly towards the gaster, with a graded drop in temperature in either direction. This article discusses possible reasons for the existence of such a hot spot in this particular location (the prothorax), its role or function, and its mode of operation. The authors speculate that it is a thermoregulatory center (for heating or cooling) that might be present in possibly all Hymenoptera that spend a considerable part of their life flying, regardless of whether they are social, parasocial, or solitary.

Diazepam inhibits reproduction and reproductive behavior of oriental hornet. A possible role for the peripheral-type benzodiazepine receptor.

Feeding of diazepam to young hornets completely inhibits or delays development of their ovaries for a relatively long period. In control hornets, the ovaries usually develop within a day or two post eclosion and comb building commences on the second day of life. The hornets then oviposit into the comb cells and the deposited ova give rise to larvae. Trials were performed on parallel groups of hornets of various ages. When the sedative diazepam was administered to hornets aged 0-24 hours the ovaries of these young hornets failed to show any development, so that no oocytes ripened and consequently there was no oviposition whatsoever. Neither were any comb cells built or, at best, only a few were built. When the diazepam was administered to hornet's being the age of 48 hours, it exerted no change, that is, the eggs developed normally and comb building was the same as in the control group. Longevity of hornets was uniform in all the test groups and similar to that in the control.

MRI of oriental hornet head.

The head of the Oriental hornet in situ, detached from a live sample was imaged using Magnetic Resonance Imaging (MRI). This non-invasive method enabled us to visualize the three-dimensional structure of the hornet's brain and intracerebral organs, as based on cubic voxels of 23 microm3. From these images, we could identify various cephalic structures in both supra-esophageal and sub-esophageal locations. In the former location, we identified and visualized the ocelli, ommatidia, mushroom body, lobula, medulla and the compound eyes in the protocerebrum, as well as the olfactory lobe and bases of the antenna in the deutocerebrum, while in the sub-esophageal region we visualized organs such as the mouthparts, the esophagus, the gnathal pouch and the salivary ducts that empty into the region. Additionally, we identified various muscles, the aorta, cuticular thickenings lending support to the interior of the head and also the cuticular skeleton providing support on the outside. All the mentioned structures and organs were visualized in their relative, normal proportions, without touching or dislocating them.

A thermoregulatory center in hornets: IR photography.

In the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae), there is on the dorsal side of the thorax, beneath the mesoscutum plate of the prothorax and around the median notal suture, a lump that, in the course of hornet activity, is warmer by 9 degrees C from the surrounding milieu and by up to 6 degrees C from other body parts of the hornet. This lump is about 1 mm in diameter, butterfly-shaped, and its upper, posterior border abuts the base of the forewings. During hornet activity and via Infra Red photography one can observe heat extensions stemming from the center of the lump and proceeding forward in the direction of the head, downward toward the legs and backwards toward the bases of the wings. The warmest region is the center of the lump, with its margins showing a lower temperature. As for the legs of the hornet, their upper part is warmer than the other parts. The temperature gradients along the hornet's body are dependent on the extent and nature of hornet activity. Thus, during flight or ventilation activity, the thorax is the warmest part of the body, while the wings, legs, and antennae, as well as the posterior part of the gaster are colder, yet all these body parts are still warmer to varying degrees than the surrounding milieu. Thus, at night, when sentry worker hornets stand guard around the nest entrance and remain practically motionless, the temperature differences between the various body parts are retained unchanged. We conjecture that the described butterfly-shaped lump is a thermoregulatory center (TC), which is neurogenically activated, since the changes occurring in it are rapid, a matter of one to several seconds and do not appear to be directly dependent on the hemolymph supply. The thermoregulatory center keeps a high constant temperature apparently related to hornet activity and the environmental conditions. The temperature cascade is most probably regulated via the tracheal system. Apparently another system activated by a heat pump mechanism keeps the gaster at a lower temperature than the environment.

Cilia in the head of hornets: form and function.

In the head of the Oriental hornet, beneath the cuticle, there are plaques of hair cells. These are distributed throughout the upper front part of the head; to wit: in the region of the vertex (i.e., around and behind the ocelli), in the genae around and behind the compound eyes (the ommatidia), and in the region of the forehead or frons. These hair cells are arranged with their thin whip-like part (i.e., cilia) directed outward and morphologically fall into three distinct groups: type (a) thin elongated cilia connected to each other alongside by side-links; type (b) thin elongated cilia of which two or more interconnect at their distal ends via a delicate nerve fiber bearing a knob at its center; and type (c) shorter and thicker cilia that roughly resemble a triangular thorn and are also interconnected by a thin thread, which, however, bears a ball rather then a knob at its center. The knob in the one case and the ball in the other vary in their diameter, but in both instances the interconnecting elements, be they nerve fibers or threads, are seemingly multidirectional. Beneath the frons, in the region of the coronal suture, the hair cells (cilial plaques) are inwardly directed and bear a large trachea at their center. Presumably, the "weighted" cilial cells that are directed toward the exterior of the body aid the hornet in navigation and gravity determination whereas the inwardly directed ciliary cells may possibly serve in acoustic communication. Another element worthy of mention within the hair cells are yellow granules (yg). These yg's originate from the whip-like portion of the ciliary cells that are distributed beneath the frons plate, and also in the yellow stripes of the gastral cuticle. Conceivably, these yellow granules, in both cases, may play a role in the absorption and storage of solar energy. In summary, ciliary structures are involved in the hornet in gravity sensing, in acoustical communication and in light sensing, i.e., with some similarity with what happens in vertebrates in the inner ear and in the photoreceptor.

Electrical properties of hornet silk: temperature dependence.

Hornet silk is a polymer of amino acids. One of the known properties of polymers is their electrical activity. The present study describes the results of electrical measurements carried out vertically on the silk cap of pupae of the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae). The measurements undertaken were the temperature-dependent electric current, voltage and resistance, all measured within the range of biological temperatures, as well as the capacitance. The temperature-dependent spontaneous current attained values up to 327 nano Amperes (nA) while the maximal voltage reached 347 millivolt (mV). The electrical resistance was low and steady (1-20 mu omega) at temperatures ranging between 19-32 degrees C, but at lower or higher temperature it increased fairly sharply by about three orders of magnitude. The electrical capacitance, computed according to the discharge curve (decay curve) amounted to 0.4 microFarad (microF). The paper also discusses the role of the pupal silk as producer of a 'clean room' while the cuticle is being laid down by the pupae after undergoing metamorphosis, as well as the significance of the measured electrical parameters vis-à-vis the developing pupae.

The effect of matrix stiffness of injectable hydrogels on the preservation of cardiac function after a heart attack.

This study compares the effect of four injectable hydrogels with different mechanical properties on the post-myocardial infarction left ventricle (LV) remodeling process. The bioactive hydrogels were synthesized from Tetronic-fibrinogen (TF) and PEG-fibrinogen (PF) conjugates; each hydrogel was supplemented with two levels of additional cross-linker to increase the matrix stiffness as measured by the shear storage modulus (G'). Infarcts created by ligating the left anterior descending coronary artery in a rodent model were treated with the hydrogels, and all four treatment groups showed an increase in wall thickness, arterial density, and viable cardiac tissue in the peri-infarct areas of the LV. Echocardiography and hemodynamics data of the PF/TF treated groups showed significant improvement of heart function associated with the attenuated effects of the remodeling process. Multi-factorial regression analysis indicated that the group with the highest modulus exhibited the best rescue of heart function and highest neovascularization. The results of this study demonstrate that multiple properties of an injectable bioactive biomaterial, and notably the matrix stiffness, provide the multifaceted stimulation necessary to preserve cardiac function and prevent adverse remodeling following a heart attack.

Enhanced infarct stabilization and neovascularization mediated by VEGF-loaded PEGylated fibrinogen hydrogel in a rodent myocardial infarction model.

Most tissue engineering therapies require biomaterials that are able to induce an angiogenic response to support tissue regeneration. In addition angiogenic growth factor signaling plays an essential role in controlling the process of angiogenesis and matrices have the potential of regulating the concentration of growth factors within the cellular microenvironment. Here we demonstrated myocardial protection and improved post-infarct vascularization of the infarcted hearts using a biosynthetic injectable hydrogel consisting of polyethylene glycol and fibrinogen (PEG-fibrinogen) loaded with vascular endothelial growth factor-A (VEGF-A). Our data revealed PEG-fibrinogen hydrogel was able to store and release VEGF-A in a sustained and controlled fashion. Upon injection after coronary artery ligation, the VEGF-loaded hydrogel significantly improved arteriogenesis and cardiac performance at 4 weeks post-infarction. The results support the future application of PEG-fibrinogen for regulating growth factor signaling in cellular microenvironment and may demonstrates a new strategy for cardiovascular repair with potential for future clinical applications.

Gravity orientation in social wasp comb cells (Vespinae) and the possible role of embedded minerals.

Social wasps and hornets maintain their nest in the dark. The building of the combs by all Vespinae is always in the direction of the gravitational force of Earth, and in each cell's ceiling, at least one 'keystone' is embedded and fastened by saliva. The sensory mechanisms that enable both building of sizeable symmetrical combs and nursing of the brood in the darkness merit investigation, and the aim of the present study was to identify and characterize the 'keystones' that exist in the ceiling and in the walls of the social wasp comb cells. Bio-ferrography was used to isolate magnetic particles on slides. These slides, as well as original cells, were analyzed in an environmental scanning electron microscope by a variety of analytical tools. It was found that both the roof and the walls of each comb cell bear minerals, like ferrites, as well as Ti and Zr. The latter two elements are less abundant in the soil around the nest. Ti and Zr are known to reflect infrared (IR) light. IR imaging showed a thermoregulatory center in the dorsal thorax of the adult Oriental hornet. It is not known yet whether these insects can sense IR light.